This grant application pertains to a critical issue in the treatment and control of parasitic diseases, the need for better chemotherapies. Amalgamating techniques of molecular biology, biochemistry, genetics, structural biology, and computational chemistry this proposal offers a multidisciplinary dissection of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani, an enzyme that renders an important, if not essential, nutritional function for the parasite, and that initiates the metabolism of allopurinol, a drug that exhibits therapeutic efficacy in both leishmaniasis and Chagas disease. These studies constitute a logical step in the implementation of a rational, structure-based strategy of drug discovery, and ultimately drug design, for the treatment and prevention of leishmaniasis and other diseases of parasitic origin. Reagents available for these studies include: i. the L. donovani, T. brucei, T. cruzi and C. fasciculata hgprt genes; ii., the L. donovani aprt gene; iii., hgprt- populations of L. donovani that were generated by targeted gene replacement;' iv., E. coli that overproduce each of the trypanosomatid HGPRTs and v., effectively unlimited amounts of L. donovani, T. brucei, T. cruzi, and C. fasciculata HGPRT proteins that appear homogeneous by SDS-PAGE. In addition, an homology-based 3-D molecular model of the L. donovani HGPRT has been computationally constructed and serves as a cornerstone for our structural studies. The first objective of this application is to evaluate the contributions of HGPRT and APRT to purine metabolism in L. donovani promastigotes by phenotypic characterization of hgprt- and aprt- null mutants that will be created by homologous gene replacement. Whether hgprt and/or aprt gene function is essential for infectivity and virulence will also be tested by generating null mutants in infective Leishmania strains. The second specific aim entails the structural characterization of the L. donovani HGPRT. The first component of this specific aim will be to evaluate the 3-D model of the protein by site-directed mutagenesis of key amino acid residues that are postulated to participate in catalytic activity or govern substrate specificity and biochemical characterization of the genetically altered proteins. The second aspect of Specific Aim II will be to introduce crystallographic methods to the structural studies for the ultimate purpose of either refining the 3-D molecular model or determining the structure of the L. donovani HGPRT protein itself. The penultimate specific aim involves the identification of key active site residues of the L. donovani HGPRT that participate in catalysis using affinity and photoaffinity labeling techniques and further evaluation of the functional role of these amino acids in catalysis by site-directed mutagenesis. Lastly, we will perform computational screens of 3-D small molecule structural databases with our molecular models, and ultimately with resolved structures, to discover novel 'lead' compounds that target the active site pocket of the L. donovani HGPRT. Computationally identified compounds from the database screens, as well as 40 procured purine base analogs, will be evaluated as potential antileishmanial compounds using a simple, yet multifaceted, screen comprising of purified recombinant HGPRT enzymes, E. coli that overexpress hgprt genes, and intact parasites.